Hematoxylin and eosin (H&E) and Masson's trichrome staining

Histological analyses were conducted on scar tissue samples from both groups. H&E staining was performed to observe cellular structures, while Masson's trichrome staining provided insights into collagen distribution. The experiments were performed according to the established protocols (18).

Briefly, slides were placed in staining can and deparaffinized by immersion in three sequential baths of absolute xylene for 4 min each. This was followed by a series of ethanol washes at concentrations of 100, 100, 95, 90 and 70%, with each wash lasting 4 min. Subsequently, the slides were rinsed under running tap water for 2 min. They were then stained with hematoxylin (cat. no. C0105, Beyotime) for 2 min, followed by another 2-min rinse under tap water. To decolorize, the slides were briefly dipped into 1% acid alcohol three times and rinsed again for 2 min. After decolorization, the slides were immersed in 2% potassium acetate for 3 min and rinsed once more for 2 min. The staining process was completed by submerging the slides in eosin for 2 min, followed by a final 2-min wash under running tap water. All of the aforementioned procedures were carried out at room temperature. The slides were then air-dried for 24 h at 38°C. Prior to observation, the slides were dipped in absolute xylene for 1 min and permanently mounted with a cover slip using DPX mounting medium (cat. no. MM1410; Shanghai Maokang Biotechnology Co., Ltd.).

Slides were deparaffinized in staining cans using three 4-min immersions in absolute xylene, followed by a descending ethanol series from 100–70% in 5% decrements, each for 4 min. They were then submerged in 60°C Bouin's solution (cat. no. HT101128; Merck KGaA) for 45 min and rinsed until clear under running tap water. Nuclei were differentiated with 8 min in modified Weigert's hematoxylin, then rinsed for 2 min. Cytoplasm and erythrocytes were stained with acid fuchsin (cat. no. HT15; Merck KGaA) for 5 min, rinsed for 2 min, treated with phosphomolybdic acid (cat. no. 51429-74-4; Merck KGaA) for 10 min, then stained with methyl blue (cat. no. 28983-56-4; Merck KGaA) for 5 min and rinsed. The slides were treated with 1% acetic acid for 1 min, dehydrated in an alcohol series from 100–70%, each for 1 min, dipped in xylene for 1 min and mounted with a cover slip using DPX (Shanghai Maokang Biotechnology Co., Ltd.). All of the aforementioned procedures were carried out at room temperature.